Protein phosphorylation provides a rapid mechanism for modulating the function of specific enzymes and regulatory proteins and is widely used as a mechanism for intracellular signaling. The second messenger, cAMP, activates a family of protein kinases that are among the best studied enzymes involved in intracellular phosphorylation. Studies in both lower eukaryotes and mammalian cells indicate that cAMP mediates the response of cells to many extracellular signals such as peptide hormones and neurotransmitters as well as providing a vehicle of intracellular regulation of cell growth and differentiation. Our research has been focused on the structure and function of the mammailian cAMP-dependent protein kinase (protein kinase A) genes. The holoenzyme of protein kinase A is composed of 2 regulatory (R) and 2 catalytic (C) subunits and is activated by the interaction of cAMP with the regulatory subunits and the release of catalytic subunits. Recent studies have demonstrated the existence of multiple genes coding for both the catalytic and regulatory subunits of protein kinase A although the functional significance of this variety of cAMP-dependent kinases is unknown. The major goals of our research are to examine the function of the multiple kinase subtypes and investigate the role of these kinases in the regulation of gene expression and cell growth. Our specific aims include: (1) characterization of the CAlpha and CBeta forms of the catalytic subunit (2) isolation of mutant forms of R and C subunits with altered functional properties (3) examination of the role of R and C subunits in the induction of specific gluconeogenic enzymes in liver and the regulation of peptide hormone synthesis and secretion in the pituitary (4) determination of the role of protein kinase A in the control of the cell cycle and response to specific oncogenes. These studies will utilize molecular genetic techniques to clone and sequence the genes coding for the kinase subunits. The tissue specific expression of R and C subunits will be examined by hybridization techniques and structural alterations in the coding sequences will be constructed by site-directed in vitro mutagenesis. The functional properties of both the native and mutated forms of R and C will be assessed by incorporation of the coding sequences into eukaryotic expression vectors and transfection into cultured mouse cells or microinjection into fertilized mouse eggs to produce transgenic mice. We expect these studies to continue to provide insight into the structure and function of this important family of protein kinases and their role in intracellular signaling mechanisms.